DE69024057T2 - TRANSFERRIN RECEPTOR-SPECIFIC ANTIBODIES - NEUROPHARMACEUTICAL MEDIUM CONJUGATE. - Google Patents

Abstract

The present invention pertains to a method for delivering a neuropharmaceutical agent across the blood brain barrier to the brain of a host. The method comprises administering to the host a therapeutically effective amount of an antibody-neuropharmaceutical agent conjugate wherein the antibody is reactive with a transferrin receptor. Other aspects of this invention include a delivery system comprising an antibody reactive with a transferrin receptor linked to a neuropharmaceutical agent and method for treating hosts afflicted with a disease associated with a neurological disorder.

Description

Basics

The capillaries that supply blood to brain tissue form the blood-brain barrier (Goldstein et al. (1986) Scientific American 255:74-83; Pardridge, W.M. (1986) Endocrin. Rev. 7:74-330). The endothelial cells of brain capillaries differ from the endothelial cells in other body tissues. The endothelial cells of brain capillaries are connected to one another via intercellular junctions that form a continuous barrier for the passive transport of substances from the blood to the brain. These cells also differ in that they have few pinocytosis vesicles, which in other tissues allow non-selective transport through the capillary wall. They also lack continuous open connections or channels running through the cells that would allow free passage.

The blood-brain barrier serves to ensure constant control of the brain environment. The levels of various substances in the blood, such as hormones, amino acids and ions, are often subject to small Fluctuations that may be induced by activities such as feeding and exercise (Goldstein et al., supra). If the brain were not protected by the blood-brain barrier from these fluctuations in serum composition, this could lead to uncontrolled neural activity.

Isolation of the brain from the bloodstream is not complete. If it were, the brain would be unable to perform its function properly due to a lack of nutrients and the lack of necessary exchange of substances with the rest of the body. The presence of specific transport systems within the capillary endothelial cells ensures that the brain receives all the substances it needs for normal growth and function in a controlled manner. In many cases, this transport system consists of membrane-bound receptors that bind their corresponding ligands and take them up into the cell (Pardridge, W.M., supra). The vesicles containing the receptor-ligand complex then migrate to the abluminal surface of the endothelial cell where the ligand is released.

The problem associated with the blood-brain barrier lies in this protective mechanism of the brain, as it excludes many potentially useful therapeutic agents. Currently, only sufficiently lipophilic substances can penetrate the blood-brain barrier (Goldstein et al, supra). Some drugs can be made more lipophilic, thereby increasing their ability to to cross the blood-brain barrier. However, each modification must be tested on a drug-by-drug basis, and the modification may result in a change in the drug's activity. The modification may also have a general effect because it increases the compound's ability to cross all cellular membranes, not just the endothelial cell membranes of brain capillaries.

EP-A-0 328 147 relates to anthracycline immunoconjugates which are specifically and subsequently taken up into specific cell types. The immunological components of these immunoconjugates are antibodies, such as anti-transferrin receptor antibodies. The immunoconjugates which may be taken up have a lethal effect on the target cells. The specific immunoconjugate is a lethal anthracycline derivative.

In one aspect, the present invention provides the use of an antibody-neuropharmaceutical agent conjugate, when the antibody reacts with a transferrin receptor, for the manufacture of a medicament for transporting the neuropharmaceutical agent across the blood-brain barrier into the brain of a patient under conditions where binding of the antibody occurs to the transferrin receptors located on endothelial cells of the brain capillaries and the neuropharmaceutical agent is transported across the blood-brain barrier in a pharmaceutically active form.

In another aspect, the invention provides a conjugate for transporting a neuropharmaceutical agent across the blood-brain barrier comprising an antibody reactive with a transferrin receptor and bound to a growth factor, whereby the conjugate transports the growth factor across the blood-brain barrier when administered in vivo.

The invention further provides a conjugate for transporting a neuropharmaceutical agent across the blood-brain barrier comprising an antibody reactive with a transferrin receptor and bound to a nucleotide analogue, whereby the conjugate transports the nucleotide analogue across the blood-brain barrier when administered in vivo. The invention further provides the use of an antibody-neuropharmaceutical agent conjugate, wherein the antibody reacts with a transferrin receptor, for the manufacture of a medicament for treating a patient suffering from a disease associated with a neurological disorder under conditions where binding of the antibody to the transferrin receptor located on an endothelial cell of the brain capillaries occurs and the neuropharmaceutical agent is transported across the blood-brain barrier in a pharmaceutically active form.

The invention further provides the use of a ligand-neuropharmaceutical agent conjugate, wherein the ligand reacts with a transferrin receptor, for Manufacture of a medicament for transporting a neuropharmaceutical agent across the blood-brain barrier to the brain of a patient under conditions where binding of the ligand occurs to transferrin receptors located on endothelial cells of brain capillaries and the neuropharmaceutical agent is transported across the blood-brain barrier in a pharmaceutically active form.

Currently available means for delivering a therapeutic agent to the brain are limited because they are invasive. The present invention is noninvasive and can utilize already available antibodies that react with a transferrin receptor as a carrier for the neuropharmaceutical agent. This is advantageous because the antibodies are able to transport a neuropharmaceutical agent across the blood-brain barrier without prematurely releasing the neuropharmaceutical agent before reaching the brain side of the blood-brain barrier. In addition, if the therapeutic activity of the agent to be delivered to the brain is not altered by adding a linker, a non-cleavable linker can be used to attach the neuropharmaceutical agent to the antibody.

Description of the drawings

Figure 1 is a graphical representation of the uptake of ¹⁴C-labeled murine monoclonal antibody (Ox-26) to the transferrin receptor in the rat brain, where the percentage of the injected dose of a radiolabeled antibody per brain and per 55 µl blood versus time after injection.

Figure 2 is a histogram showing the time-dependent changes in the distribution of radiolabeled OX-26 between brain parenchyma and vasculature.

Figure 3 is a histogram showing the enhanced transport of methotrexate across the blood-brain barrier when administered as a conjugate with OX-26.

Figure 4 shows in three histograms (A, B and C) both the distribution of the antibody and the AZT components of an OX-26-AZT conjugate in the brain.

Figure 5 is a histogram showing the experimental results of transport of a protein, namely horseradish peroxidase, across the blood-brain barrier in rat brains as a conjugate with OX-26.

Figure 6 is a histogram showing the experimental results of the transport of soluble CD4 into the rat brain parenchyma when CD4 is used as a conjugate with OX-26.

Figure 7 is a histogram showing the biodistribution of antibody 128.1 and control IgG in a macaque.

Precise description

The method for transporting a neuropharmaceutical agent across the blood-brain barrier into the brain of a patient comprises administering a therapeutically effective amount of an antibody-neuropharmaceutical The patient is administered a drug conjugate in which the antibody reacts with the transferrin receptor located on the endothelial cells of the brain capillaries. The conditions of the procedure are selected so that the antibody binds to the transferrin receptor on the endothelial cells of the brain capillaries and the neuropharmaceutical drug is transported across the blood-brain barrier in a pharmaceutically active form.

The patient may be an animal that may suffer from a neurological disease (e.g., an animal with a brain). Examples of patients include mammals, such as humans, pets (e.g., dogs, cats, cows, or horses), mice, and rats.

The neuropharmaceutical agent may be an agent having a therapeutic or prophylactic effect on a neurological disease or any physical condition affecting biological functions of the central nervous system. Examples of neurological diseases include cancer (e.g. brain tumors), autoimmune deficiency syndrome (AIDS), epilepsy, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, trauma, depression, Alzheimer's disease, migraine, pain or epileptic seizures. The classes of neuropharmaceutical agents useful in this invention include proteins, antibiotics, adrenergic agents, anticonvulsants, small molecules, nucleotide analogues, chemotherapeutics, antitrauma agents, peptides and other classes of agents used to Treatment or prevention of neurological diseases. Examples of proteins include CD4 (including soluble components thereof), growth factors (e.g. nerve growth factor and interferon), dopamine decarboxylase and tricosanthin). Examples of antibiotics include amphotericin B, gentamicin sulfate and pyrimethamine. Examples of adrenergic agents (including blockers) include dopamine and atenolol. Examples of chemotherapeutic agents include adriamycin, methotrexate, cyclophosphamide, etoposide and carboplatin. An example of an anticonvulsant that could be used is valproate and an antitrauma agent that could be used is superoxide dismutase. Examples of peptides could be somatostatin analogues and enkephalinase inhibitors. Useful nucleotide analogues include azidothymidine (hereinafter AZT), dideoxymosin (ddI) and dideoxycytidine (ddC).

Serum transferrin is a monomeric glycoprotein with a molecular weight of 80,000 daltons that binds iron in the bloodstream and transports it to the various tissues (Aisen et al. (1980) Ann. Rev. Biochem. 49:357-393; Macgillivray et al. (1981) J. Biol. Chem. 258:3543-3553). Iron uptake by individual cells is mediated by the transferrin receptor, an integral membrane glycoprotein consisting of two identical 95,000 dalton subunits linked by a disulfide bridge. The number of receptors on the cell surface appears to vary with the

cell proliferation, with the number being highest on actively growing cells and lowest on resting and differentiated cells. Jeffries et al. (Nature Vol. 312 (November 1984) pages 167-168) used monoclonal antibodies to show that brain capillary endothelial cells have transferrin receptors on their cell surface.

The antibodies usable in this invention react with a transferrin receptor. The term antibody here includes both polyclonal and monoclonal antibodies. Monoclonal antibodies that react with a transferrin receptor are preferred. The term antibody also includes mixtures of more than one antibody that reacts with a transferrin receptor (e.g. a cocktail of different monoclonal antibody types that react with a transferrin receptor). The term antibody also includes complete antibodies, biologically functional fragments thereof, chimeric antibodies that have components of more than one species, bifunctional antibodies, etc. Biologically functional antibody fragments that can be used are those fragments that are sufficient to enable the antibody fragment to bind to the transferrin receptor.

The chimeric antibodies may consist of parts derived from two different species (e.g. a human constant region and a human binding region). The chimeric antibodies may consist of parts derived from two different species (e.g. a human constant region and a human binding region). Parts can be chemically linked together using conventional techniques or can be produced as a single, continuous protein using genetic engineering techniques. DNA encoding the proteins of the two parts of the chimeric antibody can be expressed as a continuous protein

The term transferrin receptor includes the entire receptor or parts thereof. Parts of the transferrin receptor include those parts that are sufficient for the receptor to successfully bind to the anti-transferrin receptor antibody.

Monoclonal antibodies which react with at least part of the transferrin receptor can be obtained (e.g. OX-26, B3/25 (Omary et al. (1980) Nature 286,888-891), T56/14 (Gatter et al. (1983) J. Clin. Path. 36,539-545; Jeffnes et al. Immunology (1985) 54:333-341) OKT-9 (Sutherland et al. (1981) Proc. Natl. Avad. Sci. USA 78:4515-4519), L5.1 (Rovera, C. (1982) Blood 59:671- 678), 5E-9 (Haynes et al. (1981) J. Immunol. 127:347- 351), RI7 217 (Trowbridge et al. Proc. Natl. Acad. Sci. USA 78:3039 (1981) and T58/30 (Omary et al. cited above) or can be produced using conventional somatic cell hybridization techniques (Köhler and Milstein (1975) Nature 256, 495-497). A non-purified or purified protein or peptide comprising at least part of the transferrin receptor can be used as an immunogen. An animal is the immunogen to obtain anti-transferrin receptor antibody-producing spleen cells. The animal species to be immunized may vary depending on the species of the monoclonal antibody desired. The antibody-producing cell is fused with an immortalized cell (e.g., myeloma cell) to form a hybridoma cell capable of secreting anti-transferrin receptor antibodies. The unfused remaining antibody-producing cells and immortalized cells are removed. Hybridoma cells producing the anti-transferrin receptor antibodies are selected using conventional techniques, and the selected anti-transferrin receptor antibody-producing hybridoma cells are cloned and cultured.

Polyclonal antibodies can be produced by immunizing an animal with a non-purified or purified protein or peptide that has at least part of the anti-transferrin receptor. The animal is maintained under conditions where it produces antibodies reactive with a transferrin receptor. The animal's blood is collected until the desired antibody titer is reached. The serum containing the polyclonal antibodies (antisera) is separated from the other blood components. The serum containing the polyclonal antibodies can optionally be further separated into specific antibody type fractions (e.g. IgG, IgM).

The neuropharmaceutical agent can be linked to the antibody using standard chemical conjugation techniques. Typically, the linkage is through an amine or a sulfhydryl group. The linkage can be a cleavable or noncleavable linkage, depending on whether the neuropharmaceutical agent is more effective when released in its native form or whether the pharmaceutical activity of the agent is retained when bound to the antibody. The decision to use a cleavable or noncleavable linker can be made without additional testing by determining the activity of the drug in both the native and linked forms, or for some drugs, the native or linked form can be determined based on known activities of the drug.

In some cases, including delivery of the proteins or peptides to the brain, release of the free protein or peptide may not be necessary if the biologically active portion of the protein or peptide is not affected by the linkage. As a result, antibody-protein or antibody-peptide conjugates can be prepared using non-cleavable linkers. Examples of such proteins or peptides include CD4, superoxide dismutase, interferon, nerve growth factor, tricosanthin, dopamine decarboxylase, somatostatin analogs and enkephalinase inhibitors. The terms used herein, such as "CD4" includes modified versions of natural molecules such as soluble CD4, truncated CD4, etc. Examples of non-cleavable linker methods useful in the invention include the carbodiimide (EDC), the sulfhydrylmaleimide, the N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP; Pharmacia) and the periodate methods. In the carbodiimide method, a water-soluble carbodiimide reacts with the carboxylic acid groups of proteins and activates the carboxyl group. The carboxyl group is attached to an amino group of the second protein. The result of this reaction is a non-cleavable amide bond between two proteins.

In the sulfhydrylmaleimide method, a sulfhydryl group is introduced to an amine group of one of the proteins using a compound such as Traut's reagent. The other protein reacts with an NHS ester (such as gamma-maleimidobutyric acid NHS ester (GMBS)) to form a maleimide derivative that reacts with the sulfhydryl groups. The two modified proteins then react to form a non-cleavable covalent bond.

SPDP is a heterofunctional cross-linking agent that introduces aliphatic thiol groups into either the monoclonal antibody or the neuropharmaceutical agent. The thiol group reacts with an amine group to form a non-cleavable bond.

Periodate coupling requires oligosaccharide groups present either in the carrier or in the protein to be transported. If these groups are present in the protein to be transported (as in horseradish peroxidase (HRP)), an active aldehyde is formed in the protein to be transported, which reacts with the amino group of the carrier. It is also possible to form active aldehyde groups from the carbohydrate groups present in the antibody molecules. These groups can then react with the amino groups of the protein to be transported and form stable conjugates. Alternatively, the periodate-oxidized antibody can react with a hydrazide derivative of a protein to be transported, which also produces a stable conjugate.

The antibody-protein conjugate can also be made as a continuous protein using genetic engineering. Genetic constructs can be made that have DNA encoding the anti-transferrin receptor antibody fused to DNA encoding the protein to be transported across the blood-brain barrier. The protein can be expressed as a continuous molecule containing both an antibody portion and a neuropharmaceutical agent portion.

Cleavable linkers can be used to link a neuropharmaceutical agent that is to be delivered to the brain, or when a non-cleavable Linker alters the activity of the neuropharmaceutical agent.

Cleavable acidic linkers include cis-aconitic acid, cis-carboxylalkadienes, cis-carboxylalkatrienes, and polymaleic anhydrides. Other cleavable linkers are linkers capable of binding to primary alcohol groups. Examples of neuropharmaceutical agents that can be linked via a cleavable bond include AZT, ddI, ddC, adriamycin, amphotericin B, pyrimethmine, valproate, methotrexate, cyclophosphamide, carboplatin, and superoxide dismutase. The noncleavable linkers generally used to link the proteins to the antibody can also be used to link other neuropharmaceutical agents to the antibody.

In addition to covalent binding, conjugates can be formed by means of non-covalent bonds, such as those that form ionic bonds, hydrogen bonds, hydrophobic interactions, etc. with bifunctional antibodies. It is important that the conjugate bond is strong enough to allow the conjugate to pass through the blood-brain barrier. The antibody-neuropharmaceutical agent conjugates can be administered orally, by subcutaneous or other injection, intravenously, intramuscularly, parenterally, transepidermally, nasally or rectally. The form in which the conjugate is administered (e.g. as capsule, tablet, solution, emulsion) depends at least in part on the route of administration.

A therapeutically effective amount of an antibody-neuropharmaceutical agent conjugate is that amount necessary to significantly reduce or eliminate symptoms associated with a particular neurological disorder. The therapeutically effective amount is determined on an individual basis and is based at least in part on the individual's body weight, the severity of the symptoms to be treated, the results sought, the specific antibody, etc. Thus, the therapeutically effective amount can be determined by the skilled artisan using such factors and simply by ordinary experimentation. Although the above description primarily refers to antibodies, any proteins that interact with the extracellular domain of the transferrin receptor, including the ligand binding site, can potentially serve as a means of transporting drugs across the blood-brain barrier. These could include, in addition to anti-transferrin receptor antibodies, transferrin, the ligand that binds to the receptor, and any transferrin derivatives that retain receptor binding activity. Indeed, any ligand that binds to the transferrin receptor could potentially be used.

The present invention is further illustrated by the following specific examples.

EXAMPLE 1

In vitro binding of murine monoclonal antibodies to human brain endothelial cells

Two murine monoclonal antibodies recognizing the human transferrin receptor, namely 83/25 and T58/30, described by Trowbridge (U.S. Patent 4,434,156, published February 28, 1984, Nature Vol. 294 pages 171-173 (1981)), both publications incorporated by reference, were tested for their ability to bind to the endothelial cells of human brain capillaries. B3/25 and T58/30 antibody-producing hybridoma cell lines obtained from the American Type Culture Collection (ATTC) in Rockville, Maryland, were grown in DMEM medium supplemented with 2.0 mM glutamine, 10.0 mM HEPES (pH 7.2), 100 µM nonessential amino acids, and 10% heat-inactivated fetal calf serum. Hybridoma cell cultures were grown in 225 cc T-flasks to produce milligram amounts of IgG antibodies. Hybridoma cell supernatants were concentrated 50-fold by vacuum dialysis and applied to a Protein-A Sepharose column using the BIORad MAPS buffer system. The purified antibody was eluted from the column, dialyzed against 0.1 M sodium phosphate (pH 8.0), concentrated and stored in aliquots at -20ºC.

The primary cultures of human brain endothelial cells were grown in flat-bottomed microtiter plates with 96 Wells were allowed to grow for five days after seeding. Cells were then fixed using 3.0% buffered formalin and the plate blocked with 1.0% bovine serum albumin (BSA) in Dulbecco's phosphate buffered cream (DPBS). Aliquots (100 µl) of 83/25 or T58/30 antibodies were then added to the wells as either culture supernatants or purified protein (antibody concentrations ranged between 1-50 µl/ml). Antibodies that had specifically bound to the fixed cells were detected using a biotin-labeled polyclonal goat anti-mouse IgG antisera followed by biotinylated horseradish peroxidase (HRP)/avidin mixture (avidin-biotin complexation technique). Positive wells were detected using a Titertek Multiscan Enzyme Linked Immunosorbent Assay (ELISA) plate reader. The results showed that both antibodies bind to human brain capillary endothelial cells, with the T58/30 antibodies showing a higher binding capacity.

The same antibodies were also tested for binding to human brain capillaries using human brain tissue sections that were freshly frozen (without fixation), cut in a cryostat (section thickness was 5-20 µm), placed on slides and fixed in acetone (10 minutes at room temperature). These sections were then stored at -20ºC until tested.

The slides containing the human brain sections were allowed to equilibrate to room temperature before use. The sections were then rinsed in DPBS and incubated in methanol containing 0.3% H2O2 to block endogenous peroxidase activity. The sections were blocked for 15 minutes in a solution containing 0.2% skim milk powder and 2.0% methylmannopyranoside. B3/25 and T58/30 antibodies, purified as described above, were added to the sections at a concentration of 5-50 µg/ml and incubated at room temperature for one to two hours. Antibodies specifically bound to the tissue were detected using the avidin-biotin complexation (ABC) technique as described above for the ELISA assay. Staining of the capillaries in human brain sections was observed with both the B3/25 and the T58/30 antibodies. The T58/30 antibody also showed binding to the white matter of the brain cortex.

EXAMPLE 2

In vitro binding of the murine monoclonal antibody OX-26 to the rat transferrin receptor

The murine antibody OX-26, which recognizes the rat transferrin receptor, has been shown to bind to brain capillary endothelial cells in vivo (Jeffries et al., supra). The murine hybridoma cell line producing the OX26 murine antibody was obtained, and the hybridoma cell line was cultured in RPMI 1640 medium supplemented with 2.0 mM glutamine and 10% heat-inactivated fetal calf serum. The antibody OX-26 was purified using the affinity chromatography method described in Example 1.

The purified antibody was tested in vitro as described for the anti-human transferrin receptor antibodies in Example 1 to determine if it would bind to the brain capillaries in fresh frozen, acetone-fixed rat brain sections. The results showed that the OX-26 anti-transferrin receptor antibody binds to the capillaries in the rat brain sections in vitro.

EXAMPLE 3

In vivo binding of the murine monoclonal antibody OX-26 to the rat transferrin receptor

Dose range

The anti-rat transferrin receptor antibody OX-26 was tested in vivo by injecting the purified antibody (purification was carried out as described in Example 1) into the tail vein of female Sprague-Dawley rats (100-150 g). Before injection, the rats were anesthetized with halothane. Samples ranging from 2.0 mg to 0.05 mg antibody/rat were injected into the tail vein in 400 µl aliquots. All doses were tested in pairs of animals. One hour after injection, the animals were sacrificed and perfused via the heart with DPBS to remove blood from the organs. Immediately following completion of perfusion, the brain was removed and immediately frozen in liquid nitrogen. The frozen brain was then sectioned in a cryostat (30-50 µm) and the sections placed on glass microscope slides. The brain sections were then air dried at room temperature for one to two hours before fixation in acetone (10 minutes at room temperature). After this treatment, the sections may be stored at -20ºC. The OX-26 antibody was localized in the brain sections using immunohistochemical procedures as described above for the in vitro experiments in Example 1. The addition of the first antibody was not necessary since it is present in the brain sections. The results demonstrated that the OX-26 antibody binds to rat brain capillary endothelial cells and that doses less than 50 µg produce detectable levels of antibody in the brain using the procedures described here. Doses greater than 0.5 mg do not appear to result in significantly increased antibody binding to endothelial cells, suggesting that antibody binding sites may be saturated. Specific binding to capillary endothelium was not detected in liver, kidney, heart, spleen, or lung.

A nonspecific antibody of the same subclass as OX-26 (IgG 2a) was also tested in vivo to show that the observed binding of OX-26 to rat brain endothelial cells was specific for the OX- 26 antibody. 0.5 mg of the control antibody was injected per rat as described above. The results show that the staining pattern observed with the OX-26 antibody is specific for this antibody.

Time course of the experiment

Since it is possible to detect the OX-26 antibody in rat brain capillaries following in vivo administration, the time course over which this binding occurs was determined. 0.5 mg of purified OX-26 antibody was used as a standard dose and brain sections from animals sacrificed 5 minutes, 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours after injection were examined for the presence of the OX-26 antibody. All doses were administered in 400 µl aliquots and two animals were tested at each time point. Samples were injected and rats were treated at the various times after injection as described in the Dose Range section above.

The results showed that the antibody OX-26 can be detected in or on the endothelial cells of rat brain capillaries at the earliest 5 minutes and at the latest 24 hours after injection. 4 and 8 hours after injection, the staining pattern of the antibody is very spotty, which suggests that the antibody has accumulated in the pericytes.

EXAMPLE 4

The use of conjugates of the murine monoclonal antibody OX-26 for the transfer of horseradish peroxidase across the blood-brain barrier

Horseradish peroxidase (HRP; 40 kD) was chosen as the compound to be delivered to the brain because it is similar in size to several therapeutic agents and can be easily detected in the brain using an enzyme assay. HRP was linked to the OX-26 antibody using a non-cleavable periodate linkage and the ability of the antibody to act as a carrier of the compound into the brain was examined. The antibody conjugate was tested in vivo to determine whether the antibody could transport the HRP into the brain.

The antibody (10 mg) was first dialyzed overnight against 0.01 M sodium bicarbonate (pH 9.0). The HRP (10 mg) was dissolved in 2.5 ml of deionized water, 0.1 M sodium periodate (160 µl) was added and the mixture was incubated for five minutes at room temperature. Ethylene glycol (250 ml) was added to the HRP solution and then incubated for a further five minutes. This solution was then dialyzed overnight against 1.0 mM sodium acetate buffer (pH 4.4). To the dialyzed OX-26 antibody (2.0 ml, 5.08 mg/ml) was added 200 µl of 1.0 M sodium bicarbonate buffer (pH 9.5) and 1.25 ml of dialyzed HRP solution. The mixture was incubated for two hours in in the dark and then 100 µl of 10 mg/ml sodium borohydride was added. The resulting mixture was then incubated in the dark for a further two hours at 4ºC. The protein was precipitated from the solution by adding an equal volume of saturated ammonium sulfate solution and redissolved in a minimal volume of water. Free antibody was removed from the solution by chromatography over a Concanavalin A-Sepharose column (a column that binds HRP and the HRP-antibody conjugate and allows the free antibody to pass). The free HRP was removed by chromatography over a Protein A-Sepharose column that binds the antibody-HRP conjugate. The final product had an HRP/antibody ratio of 4/1.

A time course experiment identical to Example 3 was performed using the antibody-HRP conjugate. The antibody-HRP conjugate (0.5 mg) was injected in a 400 µl aliquot/rat. The animals were sacrificed at various times after injection and the brains were treated as in Example 3 above. The antibody-HRP conjugate was localized in the brain either by immunohistochemical antibody staining as in Example 3 or by direct staining of the brain sections for the presence of HRP. For HRP detection, the slides were first allowed to warm to room temperature for 30 minutes before incubation in methanol. The brain sections were then washed in DPBS and treated with 3,3-diaminobenzidine (DAB), the Substrate for HRP. The results showed that the OX-26 antibody-HRP conjugate binds to rat brain capillary endothelial cells as well as the unconjugated antibody. The punctate staining seen 4-8 hours after injection of the antibody alone was also observed with the antibody conjugate, suggesting that the conjugate can also enter the pericytes on the abluminal side of the blood-brain barrier. Taken together, these results demonstrate that the OX-26 antibody can transport a protein of at least 40 KD into the brain.

EXAMPLE 5

In vivo transport of adriamycin into the brain using the murine monoclonal antibody OX-26

A non-cleavable linker system similar to that in Example 4 was used to couple the chemotherapeutic agent Adriamycin to the antibody OX-26. The availability of antibodies that can detect Adriamycin in the same way as the method for detecting the carrier antibody described previously in Example 1 allowed the use of immunohistochemical methods to monitor the localization of the carrier antibody and the transport of Adiamycin into the brain.

To conjugate adriamycin to the antibody, the drug (10 mg in 0.5 ml DPBS) was oxidized by adding 200 µl 0.1 M sodium periodate. The mixture was incubated in the dark for one hour at room temperature. The reaction was stopped by adding 200 µl of ethylene glycol followed by incubation for five minutes. The OX-26 antibody (5.0 mg in 0.5 ml carbonate buffer (pH 9.5)) was added to the oxidized Adriamycin and incubated for one hour at room temperature. Sodium borohydride (100 ml, 10 mg/ml) was added and the mixture was incubated for an additional two hours at room temperature. The free Adriamycin was separated from the OX-26 antibody-Adriamycin conjugate by chromatography on a PD-10 column. The Adriamycin/OX-26 antibody ratio of the conjugate was 2/1 for this particular batch of the conjugate.

The effectiveness of the OX-26 antibody as a carrier for transporting Adriamycin into the brain was determined by administering 0.5 mg of the antibody-Adriamycin conjugate by injecting a 400 µl aliquot per rat into the tail vein. One hour after injection, the rat was sacrificed and the brain treated as in Example 1. All injections were performed in duplicate. As a control, 400 µg of free Adriamycin in a 400 µl aliquot was also injected into a rat. Immunohistochemical methods detected both the carrier Ox-26 antibody and Adriamycin in the rat brain sections. For Adriamycin, polyclonal rabbit anti-Adriamycin antisera and then a biotinylated goat anti-rabbit IgG antisera were applied to the sections. This was followed by the addition of a biotinylated HRP/avidin mixture and the enzymatic detection of HRP.

The results demonstrate that both the OX-26 antibody and the conjugated adriamycin are localized in rat brain capillary endothelial cells following in vivo administration. There is no evidence that free adiamycin binds to brain capillary endothelial cells or enters the brain.

Adriamycin-Ox-26 conjugate coupled through a carbodiimide bond was also synthesized (drug/antibody ratio 10/1) and tested in vivo. The results of this experiment were essentially identical to those obtained with the periodate-linked antibody-drug conjugate. In both cases, staining to the carrier antibody was very pronounced and visible in the capillaries of all brain regions. This staining was even uniform along the capillaries. Staining to adriamycin was less intense but also visible throughout the brain capillaries. Punctate staining was observed, suggesting accumulation in the pericytes located on the brain side of the blood-brain barrier.

EXAMPLE 6

In vivo transport of methotrexate into the brain using the murine monoclonal antibody OX-26

A non-cleavable carbodiimide linkage was used to attach methotrexate to the murine monoclonal antibody OX-26 A procedure analogous to that described in Example was used to control the transport of both methotrexate and the carrier antibody into the brain capillary endothelial cells.

Methotrexate was coupled to the murine monoclonal antibody OX-26 via its active ester. 81 mg (0.178 mM) methotrexate (Aldrich) was stirred with 21 mg (0.182 mM) N-hydroxysuccinimide (Aldrich) in 3 ml dimethylformamide (DMF) at 4ºC. Ethyl-3-dimethylaminopropylcarbodiimide (180 mg; EDC; 0.52 mM) was added to this solution and the reaction mixture was stirred overnight. The crude ester was purified from reaction byproducts by flash chromatography on silica gel 60 (Merck) using a solution of 10% methanol in chloroform as eluent. The purified active ester fractions were collected and concentrated to dryness. The ester was dissolved in 1 mL of DMF and stored at -20°C until use. 50 mg (50%) of active ester was recovered, as determined by an A372 (e372=7200).

An OX-26 solution containing 2.1 mg (14 nmol) of antibody in 0.9 ml of 0.1 M phosphate (pH 8.0) was thawed at 4ºC. To this stirred antibody solution was added 1.4 µl (140 nmol) of active ester prepared as described above. After 16 hours at 4ºC, the mixture was chromatographed on a Sephadex PD-10 column (Pharmacia) using phosphate buffered cream (PBS). to separate the conjugate from the free drug. Fractions containing the antibody-methotrexate conjugate were pooled. Antibody and drug concentrations were determined spectrophotometrically as described by Endo et al. (Cancer Research (1988) 48:3330-3335). The final conjugate contained 7 methotrexate molecules/antibody.

The ability of the monoclonal antibody OX-26 to transport methotrexate to the capillary endothelial cells of the rat brain was tested in vivo by injecting 0.2 mg of conjugate (in 400 µl) into the tail vein of two rats each. The animals were sacrificed one hour after injection and the brains were treated immunohistochemically as in Example 1. To detect methotrexate in the brain, a rabbit antisera directed against methotrexate was used as the first antibody. A biotinylated goat anti-rabbit antisera together with a mixture of biotinylated HRP and avidin was then used to visualize the methotrexate in the rat brain. The carrier antibody was detected as previously described.

The results of these experiments show that methotrexate as a conjugate with OX-26 accumulates along or in the capillary endothelial cells of the brain. The observed staining of methotrexate is comparable in intensity to the staining observed for the vehicle. The staining appears to occur in all brain regions and is even uniform along the capillaries.

EXAMPLE 7

Antibody fragments

The Fc portion of the murine monoclonal antibody OX-26 was removed to determine whether this would alter its localization or uptake into rat brain capillary endothelial cells. F(ab)2 fragments of OX-26 were prepared from intact Iggs by pepsin digestion. A kit from Pierce Chemical Co. contains the reagents and protocols for cleaving the antibody and obtaining the fragments. The F(ab')2 fragment (0.2 mg doses) was injected in 400 µl aliquots into the tail vein of rats. The same experiment was performed as with the intact antibody (Example 2). The F(ab')2 Fragment was detected immunohistochemically using a goat anti-mouse F(ab')2 antisera followed by the addition of biotinylated rabbit anti-goat IgG antisera. A biotinylated HRP/avidin mixture was added and the antibody complex was visualized using an HRP enzyme assay. The results showed that the F(ab)2 fragment of the OX-26 antibody binds to the capillary endothelial cells of the rat brain.

EXAMPLE 8

Measurement of OX-26 in brain tissue

To quantitatively determine the amount of OX-26 accumulated in the brain, the radioactively labelled antibody into the tail vein of rats. Antibodies were labeled with either 14C-acetate anhydride or 3H-succinimidyl propionate, essentially as described by Kummer, U., Methods in Enzvmoloav, 121: 670-678 (1986), Mondelaro, RC, and Rueckert, RR, J. of Bioloqical Chemistrv, 250:1430-1421 (1975), included here by reference. In all experiments, radiolabeled compounds were injected as a 400 µl bolus into the tail vein of halothane-anesthetized female Sprague-Dawley rats (100-125 gr), and animals were sacrificed with a lethal dose of anesthetic at the appropriate time points after injection. A 3H-labeled IgG2a control antibody served as a control for nonspecific radioactivity in the brain due to residual blood and was injected along with the 14C-labeled OX-26. At the appropriate time points after injection, animals were sacrificed and brains immediately removed and homogenized in 5 ml of 0.5% sodium dodecyl sulfate using an Omni-Mixer. An aliquot of the homogenate was incubated overnight with 2 ml of Soluene 350 tissue solubilizer prior to liquid scintillation counting. All data were obtained as disintegrations per minute (dpm). Blood samples were centrifuged to sediment red blood cells (which showed no significant binding of radioactive material) and radioactivity in a serum aliquot was determined by liquid scintillation counting.

The amount of antibody bound in the brain was determined at various times after injection to study the pharmacokinetics of brain uptake. In addition, the amount of labeled antibody in the blood was measured so that the bloodstream clearance rate could be determined. This measurement result was also used to calculate the amount of radioactivity in the brain that was attributable to blood contamination and was then subtracted from the total amount to obtain the amount of antibody specifically bound in the brain.

A maximum of 14C-labeled OX-26, corresponding to approximately 0.9% of the injected dose, was reached in the brain 1 to 4 hours after injection, as shown in Figure 1 (values are shown as mean plus or minus standard error of measurement (SEM) with N=3 rats per time point). The amount of radioactivity bound in the brain decreased steadily from 4 to 48 hours after injection to a constant level of approximately 0.3% of the injected dose. Accumulation of OX-26 in the brain was significantly reduced by the addition of an unlabeled monoclonal antibody (0.5 or 2.0 mg in the bolus injection). As an additional control, a 3H-IgG2a control antibody was injected together with the 14C-OX-26. The control antibody did not accumulate in the brain and represented the blood contamination of the brain.

In contrast to brain levels, blood OX-26 levels declined very rapidly after injection, such that one hour after injection, the percentage of the injected dose in 55 µl of blood (the blood volume bound in the brain) was approximately 0.16%, as shown in Figure 1. This is consistent with approximately 20% of the injected dose in the rat's total blood volume. Extraction of total IgG from serum and subsequent polyacrylamide gel electrophoresis (PAGE) and autoradiography showed no detectable amounts of OX-26 degradation, indicating that the antibody remains intact in the blood for 48 hours.

EXAMPLE 9

Distribution of OX-26 in brain parenchyma and capillaries

To demonstrate that anti-transferrin receptor antibody accumulates in brain parenchyma, homogenates of brains removed from animals injected with labeled OX-26 were separated by centrifugation through dextran, leaving the brain tissue in the supernatant and the capillaries as a pellet. Experiments with separated capillaries were performed according to the method of Triguero, et al., J. of Neurochemistry, 54:1882-1888 (1990), cited here as a reference. As in the uptake experiments in Example 8, the radiolabeled compounds were administered as a 400 µl bolus. injected into the tail vein of halothane-anesthetized female Sprague-Dawley rats (100-125 g), and the animals were sacrificed with a lethal dose of anesthetic at appropriate times after injection. A ³H-labeled IgG2a control antibody was injected along with the ¹⁴C-labeled OX-26 as a control for nonspecific radioactivity due to residual blood in the brain. After sacrifice, the brains were removed and kept on ice. After initial grinding, the brains were manually homogenized (8-10 strokes) in 3.5 ml of ice-cold physiological buffer (100 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 14.5 mM HEPES, 10 mM D-glucose, pH 7.4). Four ml of 26% dextran solution in buffer was added and homogenization continued (3 strokes). After removing an aliquot of homogenate, the remainder was centrifuged in a rotor with swing-out cups at 7200 rpm. The resulting supernatant was carefully removed from the capillary sediment. The entire capillary sediment and aliquots of homogenate and residue were incubated overnight with 2 ml Soluene 350 prior to liquid scintillation counting. This procedure removes more than 90% of the vessels from the brain homogenate (Triguero et al., supra).

A comparison of the relative amounts of radioactivity in the different brain fractions as a function of time shows whether transcytosis of the labelled antibody has occurred. The amount of OX-26 in the total Brain homogenate, in the brain parenchymal fraction, and in the brain capillary fraction at early (30 minutes) and late (24 hours) time points after injection is shown in Figure 2. Values in Figure 2 are presented as mean +/- SEM with N3 rats per time point. After 30 minutes, more radiolabeled antibody is bound in the capillary fraction than in the brain parenchymal fraction (0.36% of the injected dose (%ID) and 0.23%ID, respectively). At 24 hours after injection, the distribution is reversed and the majority of the radioactivity (0.36%ID) is in the parenchymal fraction compared to the capillary fraction (0.12%ID). The redistribution of radiolabeled OX-26 from the capillary fraction to the parenchymal fraction is consistent with time-dependent migration of anti-transferrin receptor antibody across the blood-brain barrier.

EXAMPLE 10

Distribution of an Ox-26-methotrexate conjugate in brain parenchyma and capillaries

Investigations of the separated capillaries were carried out according to the procedure described in Example 9 with an OX-26-methotrexate (MTX) conjugate linked via a gamma-hydrazide where the MTX part was labelled with ³H, see Kralovec, et al., J. of Medicinal Chem., 32:2426-2431 (1989), incorporated herein by reference. As with an unconjugated antibody, the labeled amount in the capillary fraction is greater than in the parenchymal fraction 30 minutes after injection (approximately twice as shown in Figure 3 with data presented as mean +/-SEM and N=3 rats per time point). This distribution changes with time so that 24 hours after injection there is approximately 4.5 times more of the labeled MTX in the brain parenchyma than in the capillaries. These results are consistent with those obtained with an unconjugated antibody and again suggest that these compounds cross the blood-brain barrier.

To be sure that these results were not due to a contaminating amount of free ³H-MTX or ³H-Mtx cleaved from the conjugate after injection, a mixture of labeled drug and antibody was co-injected into rats and an examination of the separated capillaries was performed. The amount of ³H-MTX in the different brain fractions is significantly lower for the co-mixture compared to the conjugate (more than 47 times as much as in the capillary fraction 30 minutes after injection, as shown in Figure 3). ³H-MTX and the co-mixture also show no changes in the label distribution between the different brain fractions over time as observed with the antibody-MTX conjugate or the antibody alone. These results indicate that the transport of ³H-MTX across the blood-brain barrier into the brain parenchyma is mediated by the conjugation of the drug to the anti-transferrin receptor antibody OX-26 is significantly increased.

EXAMPLE 11

Distribution of OX-26-AZT in brain parenchyma and capillaries

Studies of the separated capillaries were carried out according to the procedure of Example 9 with an OX-26-AZT conjugate using a pH-sensitive succinate linker. In these studies, a double-labeled conjugate was used where AZT was 14C-labeled and the carrier antibody was 3H-labeled. The use of such conjugates allowed independent monitoring of the distribution of both the antibody and AZT within the brain.

The linker was synthesized as follows: Succinic anhydride was used to acylate AZT by reacting equimolar amounts of these two compounds for 3 hours at room temperature under argon in the presence of dimethylaminopyridine and sodium bisulfate in freshly distilled pyridine. The product was isolated by chromatography on a DEAE Sephadex A50 column using triethylammonium bicarbonate buffer as eluent. The succinate derivative of AZT was activated at the carboxyl group as an NHS ester by reacting equimolar amounts of N-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) in freshly distilled THF for 2 hours at 4ºC. The product was purified by flash Purified by chromatography on silica gel. The resulting NHS ester of AZT succinate was used to acylate amine groups of OX-26 to form an AXT-OX-26 conjugate. A 15-fold molar excess of AZT-OX-26 NHS ester was allowed to react with OX-26 in HEPES buffer overnight at 4°C. The antibody-drug conjugate was separated from the free drug on a PD-10 column. The molar ratio of drug to antibody was 7:1. In these studies, a double-labeled conjugate was used where the AZT was 14C-labeled and the carrier antibody was 3H-labeled.

Similar levels of OX-26 and AZT were observed in the capillary fraction of the brain, and these levels decreased over time, suggesting that the substances are not retained by the capillary endothelial cells, as shown in Figure 4c. As Ox-26 levels decrease in the capillary fraction, they increase in the parenchymal fraction, indicating that the antibody migrates from the capillaries into the parenchyma in a time-dependent manner, as shown in Figure 4b. In contrast, AZT levels do not increase significantly in the brain parenchyma, suggesting that the majority of the drug is released in the endothelial cells and is not transported across the blood-brain barrier. OX-26 and AZT levels remained similar in the unfractionated homogenates over the time shown in Figure 4a. Data in Figure 4a are expressed as mean +/- SEM with N = 3 rats per time point. These results indicate that the linker was cleaved within the endothelial cells and may represent a method for delivering compounds into such cells.

EXAMPLE 12

Distribution of OX-26 horseradish peroxidase (HRP) in brain parenchyma and capillaries

Studies of the cleaved capillaries were performed using the procedures previously described for OX-26 in Example 9 and were performed with a ³H-labeled OX-26-HRP conjugate prepared using a noncleavable periodate linkage described in Example 4. Both the antibody and the HRP portion of the conjugate were labeled with tritium. One hour after injection, the majority of the radioactivity bound to the brain is in the capillary fraction, as shown in Figure 5. The data in Figure 5 are presented as mean +/- SEM with N = 3 rats per time point. Four hours after injection, the distribution of the radioactivity bound to the brain changed so that the majority is in the brain parenchymal fraction. 24 hours after injection, essentially all of the 3H-labeled Ox-26-HRP conjugate is in the parenchymal fraction of the brain, indicating that the substance has crossed the blood-brain barrier. Similar results were obtained in experiments in which only the HRP portion of the conjugate was radiolabeled.

The percentage of the injected dose of OX-26-HRP conjugate that reaches the brain is somewhat less than that of the antibody alone or the Ox-26-MTX conjugate. This is most likely due to the presence of two to three 40 kD molecules bound to each carrier and that these "passenger" molecules are randomly bound to the carrier. As a result, many of the HRP passengers may be bound to the antibody in such a way that they interfere with antigen recognition. This problem can be reduced by appropriately controlling passenger binding to regions of the carrier remote from critical functional domains.

EXAMPLE 13

Distribution of OX-26-CD4 in brain parenchyma and capillaries

A soluble form of CD4 consisting of amino acids 1-368 was linked to OX-26 by means of a linkage that directed the binding of CD4 to the carbohydrate groups located in the Fc portion of the antibody. By controlling the binding site in this way, the likelihood of the passenger molecule interfering with the antibody-antigen recognition site is reduced. Binding of the proteins to each other was achieved by a prior introduction of a sulfhydryl group into CD4 using SATA (N-succinimidyl-S-acetylthioacetate), a commercially available compound. A

Hydricide derivative of SDPD, another commercially available cross-linking reagent, was linked to OX-26 via the carbohydrate groups of the antibody. The reaction of the two modified proteins leads to a disulfide-linked conjugate.

More specifically, the linkage between the proteins was achieved by a preliminary introduction of a sulfhydryl group into CD4 using N-succinimidyl-S-acetylthioacetate (SATA), a commercially available compound. A 4-fold molar excess of SATA was added to 5 mg of CD4 in 0.1 M sodium phosphate buffer containing 3 mM EDTA (pH 7.5). This mixture was allowed to react in the dark at room temperature for 30 minutes. The unreacted starting compounds were removed by passage through a PD-10 column. A hydrazide derivative of SPDP, another commercially available cross-linking reagent, was linked to OX-26 through carbohydrate groups of the antibody. Ten milligrams of OX-26 in 2.0 ml of 0.1 M sodium acetate, 0.15 M sodium chloride (pH 5.0) was reacted with a 100-fold molar excess of sodium periodate for one hour at 4ºC in the dark. The unreacted starting materials were removed by running over a PD-10 column. The oxidized antibody was reacted with a 30-fold molar excess of hydrazide-SPDP overnight at 4ºC with stirring. The reaction of the two modified proteins leads to a disulfide-linked conjugate. One tenth volume of 0.5 M hydroxylamine was added to the thioacetylated CD4 (CD4-DATA) and the derivatized antibody was then added so that the ratio of CD4 to antibody was 7.5:1. This mixture was allowed to react at room temperature for 2 hours in the dark. The conjugate was purified by running the reaction mixture over a protein A column and then over a CD4 affinity column.

Experiments with separated capillaries and OX-26 were performed following the procedures described in Example 9 and were carried out with an Ox-26-CD4 conjugate where only the CD4 portion was 3H-labeled. Time-dependent changes in the distribution of the labeled conjugate between the capillary and parenchymal fractions of the brain consistent with transcytosis across the blood-brain barrier were observed as shown in Figure 6. The data in Figure 6 are presented as mean +/- SEM with N = 3 rats per time point.

EXAMPLE 14

Biodistribution and uptake of anti-human transferrin receptor antibodies into the brain of macaque monkeys

A panel of 32 murine monoclonal antibodies recognizing various epitopes on the human transferrin receptor were tested for reactivity with capillary endothelial cells in human, macaque, rat and rabbit brain sections by immunohistochemical procedures described in Example 1. We obtained these antibodies from Dr. Ian Trowbridge, Salk Institute, La Jolla, CA. All 32 antibodies showed low reactivity with human brain endothelial cells. Two antibodies reacted very weakly with rabbit brain capillaries and none reacted with rat capillaries. While 21 of the antibodies reacted with monkey brain capillaries, only two showed strong reactivity comparable to the observed reactivity with human brain capillaries. These 2 antibodies are referred to here as 128.1 and 335.2.

These antibodies were used to determine the tissue distribution and blood clearance of 14C-labeled anti-human transferrin receptor antibodies 128.1 and 335.2 in 2 male macaques. 128.1 and 335.2 were administered simultaneously with a 3H-labeled control IgG to one of the monkeys by intravenous catheter. During the course of the experiment, blood samples were collected to determine blood clearance of the antibodies from the bloodstream. 24 hours after injection, the animals were sacrificed and selected organs and characteristic tissues were collected by pyrolysis to determine isotope distribution and clearance. In addition, samples from various brain regions were treated as described for the separated capillary experiments in Example 9 to determine whether the antibodies had passed the blood-brain barrier. The results of the separated capillary experiments were obtained from samples of cortex, frontal cortex, cerebellum and striatum. In all samples, more than 90% of the antibodies 128.1 and 335.2 were in the brain parenchyma, suggesting that the antibodies crossed the blood-brain barrier. Control antibody levels were 5 to 10 times lower in the same samples. Using the average brain homogenate value in dpM/g tissue, the percentage of the injected dose of 128.1 in the whole brain is approximately 0.2-0.3%. This is comparable to a value of 0.3-0.5% for OX-26 in rats 24 hours after injection. A comparison of the ratios of 128.1 to control antibody for various organs is shown in Figure 7. Similar results were also obtained for 335.2. These results suggest that 128.1 is preferentially taken up by the brain compared to the control antibody. For most organs and tissues tested, the ratio of 128.1 to control is less than 2.

Claims (11)

1. Use of an antibody-neuropharmaceutical agent conjugate when the antibody reacts with a transferrin receptor for the manufacture of a medicament for transporting the neuropharmaceutical agent across the blood-brain barrier to the brain of a patient under conditions where binding of the antibody occurs to transferrin receptors located on endothelial cells of brain capillaries and the neuropharmaceutical agent is transported across the blood-brain barrier in a pharmaceutically active form. 2. Use according to claim 1, wherein the neuropharmaceutical agent is (a) a protein, e.g. CD4, tricosanthin, interferon, a nerve growth factor or dopamine decarboxylase; or (b) an antibiotic, e.g. amphotericin B, gentamycin or pyrimethamine; or (c) an adrenergic agent, e.g. dopamine or atenolol; or (d) a nucleotide analogue, e.g. azidothymidine, dideoxymosin or dideoxycytodine; or (e) a chemotherapeutic agent selected from e.g. methotrexate, cyclophosphamide, etoposide, carboplatin and adriamycin; or (f) an anticonvulsant, e.g. valproate; or (g) an antitrauma agent, e.g. B. superoxide dismutase; or (h) a peptide, e.g. a somatotostatin analogue or an enkephalinase inhibitor. 3. Use according to claim 1, wherein the antibody is a monoclonal antibody selected from e.g. OX-26, B3/25, T56/14, OKT9, L5.1, 5E-9, R17 217 and T58/30. 4. Use according to claim 1, wherein the neuropharmaceutical agent is linked to the antibody via a cleavable bond, and wherein, for example, the cleavable bond is formed by using cis-aconitic acid, cis-carboxylalkatriene or polymaleic anhydride. 5. Use according to claim 4, wherein the neuropharmaceutical agent is selected from azidothymidine, dideoxymosin, dideoxycytodine, adriamycin, amphotericin B, pyrimethamine, valproate, methotrexate, cyclophosphamide, carboplatin, gentamycin, dopamine, atenolol and etoposide. 6. Use according to claim 1, wherein the neuropharmaceutical agent is linked to the antibody via a non-cleavable bond, e.g. an amide bond, a bond between a sulfhydryl group and a maleimide derivative, a bond between a thiol and an amino group, or a bond between an aldehyde and an amino group. 7. Use according to claim 1, wherein the neuropharmaceutical agent is a protein or peptide. 8. A conjugate comprising an antibody reactive with a transferrin receptor and bound to a growth factor as the neuropharmaceutical agent for transporting a neuropharmaceutical agent across the blood-brain barrier, whereby the conjugate transports the growth factor, a neuropharmaceutical agent, across the blood-brain barrier when administered in vivo. 9. A conjugate comprising an antibody reactive with a transferrin receptor and bound to a nucleotide analogue as the pharmaceutical agent for transporting a neuropharmaceutical agent across the blood-brain barrier, whereby the conjugate transports the nucleotide analogue, a pharmaceutical agent, across the blood-brain barrier when administered in vivo. 10. Use of an antibody-neuropharmaceutical agent conjugate, wherein the antibody reacts with a transferrin receptor, for the manufacture of a medicament for treating a patient suffering from a disease associated with a neurological disorder under conditions where binding of the antibody occurs to the transferrin receptor located on an endothelial cell of the brain capillaries and the neuropharmaceutical agent is transported across the blood-brain barrier in a pharmaceutically active form. 11. Use of a ligand-pharmaceutical agent conjugate, wherein the ligand reacts with a transferrin receptor, for the manufacture of a medicament for transporting a neuropharmaceutical agent across the blood-brain barrier to the brain of a patient under conditions where binding of the ligand occurs to transferrin receptors located on endothelial cells of brain capillaries and the neuropharmaceutical agent is transported across the blood-brain barrier in a pharmaceutically active form.